阅读说明：本技术 一种抗念珠菌甘露聚糖的兔源单克隆抗体及其应用 (Rabbit-derived monoclonal antibody for resisting candida mannan and application thereof ) 是由 刘春龙 付成华 翟栓柱 盛长忠 周泽奇 粟艳 于 2019-12-17 设计创作，主要内容包括：本发明提供了一种抗念珠菌甘露聚糖的单克隆抗体及其应用,所述单克隆抗体的重链可变区包括如SEQ ID NO:7所示的氨基酸序列；所述单克隆抗体的轻链可变区包括如SEQ ID NO:8所示的氨基酸序列。本发明的单克隆抗体从新西兰大耳兔内获得,对念珠菌甘露聚糖具有较强的结合活性和中和活性,稳定性好,可潜在地应用于念珠菌感染的临床样本的检测与鉴定以及念珠菌抑制剂研发等。(The invention provides a monoclonal antibody of anti-candida mannan and application thereof, wherein a heavy chain variable region of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 7; the variable region of the light chain of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 8. The monoclonal antibody is obtained from New Zealand big ear rabbits, has strong binding activity and neutralization activity on candida mannan, has good stability, and can be potentially applied to detection and identification of clinical samples infected by candida, research and development of candida inhibitors and the like.)

1. An antigen-binding fragment, wherein the heavy chain variable region of the antigen-binding fragment comprises the heavy chain CDR3 as set forth in SEQ id No. 3;

the light chain variable region of the antigen-binding fragment includes the light chain CDR3 shown in SEQ ID NO. 6.

2. The antigen-binding fragment of claim 1, wherein the heavy chain variable region of the antigen-binding fragment further comprises heavy chain CDR2 shown in SEQ ID NO. 2, heavy chain CDR1 shown in SEQ ID NO. 1;

the light chain variable region of the antigen binding fragment further includes light chain CDR2 shown in SEQ ID NO. 5 and light chain CDR1 shown in SEQ ID NO. 4.

3. A monoclonal antibody against candida mannan, comprising the antigen binding fragment of claim 1 or 2;

preferably, the heavy chain variable region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 7;

the variable region of the light chain of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 8;

preferably, the monoclonal antibody further comprises any one of or a combination of at least two of rabbit IgG1, IgG2, IgG3, or IgG4 constant regions, preferably rabbit IgG1 constant regions.

4. A hybridoma cell that produces the monoclonal antibody of claim 3.

5. A nucleic acid molecule comprising a DNA fragment encoding the antigen-binding fragment of claim 1 or 2 and/or the heavy chain variable region and/or the light chain variable region of the monoclonal antibody of claim 3;

preferably, the heavy chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in SEQ ID NO 9;

preferably, the light chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in SEQ ID NO 10.

6. An expression vector comprising the nucleic acid molecule of claim 5;

preferably, the expression vector further comprises a nucleic acid molecule encoding the constant region of rabbit IgG 1.

7. A host cell transfected with the nucleic acid molecule of claim 5 and/or the expression vector of claim 6;

preferably, the host cell comprises a 293T cell or a CHO cell.

8. A pharmaceutical composition comprising any one of the antigen-binding fragment of claim 1 or 2, the monoclonal antibody of claim 3, the hybridoma cell of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7, or a combination of at least two thereof;

preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

9. A kit comprising any one of, or a combination of at least two of, the antigen-binding fragment of claim 1 or 2, the monoclonal antibody of claim 3, the hybridoma cell of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, or the host cell of claim 7;

preferably, the kit further comprises any one or combination of at least two of a positive control substance, a negative control substance, an antibody diluent, a developing solution, a stop solution, a blocking solution or a washing solution.

10. Use of the antigen-binding fragment of claim 1 or 2, the monoclonal antibody of claim 3, the hybridoma cell of claim 4, the nucleic acid molecule of claim 5, the expression vector of claim 6, the host cell of claim 7, the pharmaceutical composition of claim 8, or the kit of claim 9 for the preparation of a therapeutic and/or detection agent for candida infectious diseases.

Technical Field

The invention belongs to the technical field of biology, and relates to a rabbit-derived monoclonal antibody for resisting candida mannan and application thereof.

Background

In recent years, there has been a significant increase in candida albicans infection, which is the 4 th position in blood borne infections and the 3 rd position in catheter-related infections. Even with antifungal drugs used during treatment, mortality rates due to candida infection are as high as 40%. Systemic candida infections are typically free of clinical symptoms and lack specific early diagnostic methods. At present, the gold standard for detecting candida infection is blood culture, however, after the candida infection, the disease condition develops rapidly, the traditional culture method has a long period and low positive detection rate, and the phenomena of missed diagnosis and misdiagnosis are often caused, and the disease condition is easily delayed. Therefore, there is a need to establish a new diagnostic method for candida infections.

Monoclonal antibodies are highly homogeneous antibodies produced by a single B cell clone and directed only to a specific epitope, and are generally prepared by using hybridoma cells, and after sensitized B cells having the ability to secrete specific antibodies and myeloma cells having an unlimited reproductive ability are fused into B cell hybridomas based on a cell fusion technique, and cultured into a cell population, specific antibodies directed to one epitope, i.e., monoclonal antibodies, can be prepared.

The antibody detection method is becoming a new diagnosis method for fungal infection due to its high detection speed and high accuracy. CN105968198A discloses a monoclonal antibody of candida mannan and a preparation method thereof, wherein the monoclonal antibody has the characteristics of good specificity, high titer and high purity, and has the advantages of high sensitivity, good specificity and wide application prospect when being used for candida detection in the fields of medical sanitation and scientific research.

Therefore, the construction of a new monoclonal antibody against candida has wide application prospect in the field of diagnosis and treatment of fungal infection.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides a rabbit-derived monoclonal antibody of anti-candida mannan and application thereof, wherein the monoclonal antibody is the rabbit-derived monoclonal antibody, has the characteristics of multiple antigen recognition sites, good specificity and high affinity, solves the problem of practical application of the mouse-derived monoclonal antibody, and provides a new scheme for establishing candida detection, diagnosis, prevention and treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides an antigen-binding fragment, the heavy chain variable region of which comprises the heavy chain CDR3 shown in SEQ ID NO. 3;

the light chain variable region of the antigen-binding fragment comprises the light chain CDR3 shown in SEQ ID NO. 6;

SEQ ID NO:3：YWSKTVMIVRQ；

SEQ ID NO:6：YTGTWYSNNNCQ。

preferably, the heavy chain variable region of the antigen-binding fragment further comprises heavy chain CDR2 shown in SEQ ID NO. 2, heavy chain CDR1 shown in SEQ ID NO. 1;

the light chain variable region of the antigen-binding fragment further comprises light chain CDR2 as set forth in SEQ ID NO. 5, light chain CDR1 as set forth in SEQ ID NO. 4;

SEQ ID NO:2：TNYYTSTTSPTTED；

SEQ ID NO:1：YTGYANDLK；

SEQ ID NO:5：GYKYDRLAATIDSQKP；

SEQ ID NO:4：QATYYTPSSSEACA。

in the invention, a new zealand big ear rabbit is selected as an experimental animal, and candida mannan is used as an antigen for immunization, so that the obtained monoclonal antibody has good specificity, strong affinity and high homology with human.

In a second aspect, the present invention provides a monoclonal antibody against candida mannan, the monoclonal antibody comprising the antigen binding fragment as described in the first aspect.

Preferably, the heavy chain variable region of the monoclonal antibody comprises the amino acid sequence shown as SEQ ID NO. 7;

the variable region of the light chain of the monoclonal antibody comprises an amino acid sequence shown as SEQ ID NO. 8;

SEQ ID NO:7：

MTLTCFCARTISKTSTRLTGLEYIGMISGATVDLKMYGMDLWGPGTLVTVSSVQCQSVEEVSGFSLSSYITNYYYVRQAPGKDMNWVTYVTPGTPLSGGNTGETGLRWLLLVAVLKGYANWTSPTTEDTANGRF；

SEQ ID NO:8：

MDTRAPTQLLGATFAIVMTQSEACAGYKYTGLLLLNRLANCQATEVVVKWLPGTWYQFTLTISDGSVPVCDQDATLASGVPSRFKGSGATYYTPSSVGGTIDSQKPGSSGTQVGDTVPPKLIAFGLIYQSVYSN。

preferably, the monoclonal antibody further comprises any one of or a combination of at least two of rabbit IgG1, IgG2, IgG3, or IgG4 constant regions, preferably rabbit IgG1 constant regions.

In a third aspect, the present invention provides a hybridoma cell which produces a monoclonal antibody as described in the second aspect.

In a fourth aspect, the present invention provides a nucleic acid molecule comprising a DNA fragment encoding the antigen-binding fragment of the first aspect and/or the heavy chain variable region and/or the light chain variable region of the monoclonal antibody of the second aspect.

Preferably, the heavy chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in SEQ ID NO 9;

SEQ ID NO:9：

ATGACTTTAACCTGTTTTTGCGCTCGTACAATTTCTAAAACGTCCACTCGCTTGACCGGTCTTGAATATATCGGCATGATATCAGGAGCCACAGTTGATCTCAAGATGTACGGGATGGACCTATGGGGTCCTGGCACGCTGGTCACTGTATCGAGTGTGCAATGTCAGAGCGTTGAGGAAGTCTCTGGATTCTCCTTATCATCGTATATTACCAATTACTATTACGTACGACAAGCACCCGGGAAAGATATGAACTGGGTGACATATGTTACGCCAGGTACTCCGTTGAGTGGCGGAAATACCGGGGAGACAGGTCTTCGGTGGCTCCTACTGGTCGCGGTATTAAAGGGCTACGCTAACTGGACGAGCCCTACTACCGAAGACACAGCCAATGGAAGATTT。

preferably, the light chain variable region of the monoclonal antibody comprises a nucleic acid molecule as set forth in SEQ ID NO 10;

SEQ ID NO:10：

ATGGATACTCGTGCTCCTACCCAATTATTGGGTGCCACATTTGCAATTGTTATGACGCAGTCTGAAGCGTGTGCTGGCTATAAATACACTGGACTTCTCCTACTGAATCGCTTAGCCAACTGCCAAGCAACCGAGGTCGTAGTGAAGTGGTTGCCCGGGACATGGTATCAGTTCACGCTTACTATCTCCGACGGTTCAGTTCCAGTCTGTGATCAAGACGCGACCCTCGCTTCGGGCGTACCGAGTCGATTTAAAGGAAGCGGGGCCACATACTATACGCCTTCTTCCGTGGGTGGCACTATAGATTCACAGAAGCCCGGATCGAGTGGGACCCAAGTTGGTGACACAGTCCCACCGAAACTAATTGCATTCGGCCTGATCTACCAGAGCGTATATTCTAAT。

in a fifth aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the fourth aspect.

Preferably, the expression vector further comprises a nucleic acid molecule encoding the constant region of rabbit IgG 1.

In a sixth aspect, the present invention provides a host cell transfected with a nucleic acid molecule according to the fourth aspect and/or an expression vector according to the fifth aspect.

Preferably, the host cell comprises a 293T cell or a CHO cell.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising any one of or a combination of at least two of the antigen-binding fragment of the first aspect, the monoclonal antibody of the second aspect, the hybridoma cell of the third aspect, the nucleic acid molecule of the fourth aspect, the expression vector of the fifth aspect or the host cell of the sixth aspect.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In an eighth aspect, the present invention provides a kit comprising any one of, or a combination of at least two of, an antigen-binding fragment according to the first aspect, a monoclonal antibody according to the second aspect, a hybridoma cell according to the third aspect, a nucleic acid molecule according to the fourth aspect, an expression vector according to the fifth aspect, or a host cell according to the sixth aspect.

Preferably, the kit further comprises any one or combination of at least two of a positive control substance, a negative control substance, an antibody diluent, a developing solution, a stop solution, a blocking solution or a washing solution.

In a ninth aspect, the present invention provides a use of the antigen binding fragment according to the first aspect, the monoclonal antibody according to the second aspect, the hybridoma cell according to the third aspect, the nucleic acid molecule according to the fourth aspect, the expression vector according to the fifth aspect, the host cell according to the sixth aspect, the pharmaceutical composition according to the seventh aspect, or the kit according to the eighth aspect, in the preparation of a drug for treating and/or detecting candida infections.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention selects New Zealand big ear rabbit as experimental animal, prepares monoclonal antibody, overcomes the weak affinity defect of mouse source antibody;

(2) according to the invention, candida mannan is used as an antigen for immunization, and the obtained spleen cells are subjected to cell fusion with myeloma cells to obtain a rabbit-derived monoclonal antibody, so that the rabbit-derived monoclonal antibody has good stability and strong affinity to the candida mannan;

(3) the monoclonal antibody prepared by the invention has good specificity and strong affinity with mannan, but does not generate cross reaction with other saccharides such as galactomannan, capsular polysaccharide, lipopolysaccharide and the like, and can be potentially applied to antigen detection of candida, detection and identification of clinical samples infected by candida and the like.

Drawings

FIG. 1 is an electrophoresis diagram of a monoclonal antibody, wherein lane M shows the molecular weight of the protein, and lane 1 shows the result of the electrophoresis of the monoclonal antibody;

FIG. 2 shows the potency assay of monoclonal antibodies against Mn;

FIG. 3 is a cross-reaction diagram.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.